In the clinical diagnosis of respiratory bacterial diseases, such as tuberculosis, a sample of sputum or other body fluid obtained from the patient is cultured in an agar growth medium to test for the presence of the particular bacterium of interest. Unfortunately, this is a relatively time-consuming process, generally requiring several days to produce a definitive result. During this time, a patient suspected of having tuberculosis, for example, must be isolated to prevent further spread of the disease.
The advent of DNA probes, which can identify a specific bacterium by testing for the presence of a unique DNA sequence in the sample obtained from the patient, has greatly increased the speed and reliability of clinical diagnostic testing. A test for the tuberculosis mycobacterium, for example, can be completed within a matter of hours using DNA probe technology. This allows treatment to begin more quickly and avoids the need for long patient isolation times.
In the use of DNA probes for clinical diagnostic purposes, a nucleic acid amplification reaction is carried out in order to multiply the target nucleic acid into millions of copies or amplicons. Examples of nucleic acid amplification reactions which can be used include strand displacement amplification (SDA) and polymerase chain reaction (PCR). Unfortunately, nucleic acid amplification reactions can become contaminated with the amplicons produced by previous amplification reactions. The contaminating amplicons can, in turn, contaminate new samples entering the lab, leading to false positive diagnoses.
Decontamination techniques have been developed in which contaminating amplicons produced by previous amplification reactions are recognized and destroyed. By carrying out a decontamination reaction prior to amplification, the possibility that a contaminating amplicon will be recognized as a target nucleic acid is greatly decreased. However, because decontamination and amplification reagents are often not compatible with each other and require their own reaction conditions, they must often be carried out in separate containers. In transferring the sample from one container to another, recontamination can occur.
In order to minimize contamination problems, separate areas of a clinical diagnostic laboratory are often reserved for sample preparation, amplification/decontamination and assay (detection). Although this is an effective safeguard, it is very labor-intensive and offsets some of the advantages offered by DNA probe techniques. Automation of all or part of the procedure would be desirable, but this is difficult to achieve when many processing steps are involved and the potential for cross-contamination between samples is great.
In the aforementioned copending patent application of Allen S. Reichler et al entitled "Nucleic Acid Amplification Method and Apparatus", a disposable, single-use apparatus or module is described which allows decontamination and amplification of a liquid biological sample to be carried out within the confines of a single container. In general, the disclosed apparatus includes a sample area for receiving a liquid biological sample, at least one reaction area in fluid communication with the sample area, a pneumatic area in pneumatic communication with the reaction area and the sample area, and a pneumatic port in the pneumatic area for allowing connection of the apparatus or module to a pneumatic aspiration/dispensing means. Operation of the pneumatic aspiration/dispensing means causes the liquid biological sample to flow between the sample area and the reaction area, and between different zones in the reaction area, in a controlled manner. Reagents necessary for the decontamination and amplification reactions are affixed to separate, discrete locations within the reaction area, and are contacted by the liquid biological sample at different times under the control of the pneumatic aspiration/dispensing means.
It is an object of the present invention to provide an automated system for carrying out reactions on a plurality of liquid samples using disposable, single-use modules of the general type described above.
It is another object of the invention to provide an automated system for carrying out reactions on a plurality of liquid samples, particularly nucleic acid based diagnostic assays, with little or no intervention by a human operator.
It is further object of the invention to provide an automated system for carrying out reactions on a plurality of liquid samples, particularly nucleic acid based diagnostic assays, while minimizing the potential for cross-contamination between different samples.
It is a still further object of the invention to provide improved methods for carrying out reactions on a plurality of liquid samples, particularly nucleic acid based diagnostic assays, which methods can be carried out using the exemplary apparatus disclosed and claimed herein.